This invention relates to novel stable salts of 5,10-methylene-N-[4-[[2-amino-1,4,5,6,7,8-hexahydro-4-oxo-(6R)-, (6R,S)-, or (6S)-pteridinyl)-methyl]amino]-benzoyl]-L-glutamic acid [hereinafter "5,10-methylene-(6R)-, (6R,S)- or (6S)-tetrahydrofolic acid"].
Tetrahydrofolates are the biologically active forms of folic acid (folic acid co-factors).
As pharmaceuticals, tetrahydrofolates are mainly used as the calcium salt of 5-formyl-5,6,7,8-tetrahydrofolic acid (leucovorin), for example, for increasing the therapeutic effect of 5-fluorouracil or, for example, as a rescue substance when using methotrexate in cancer therapy.
In the body, 5-formyl-(6S)-tetrahydrofolic acid is converted to 5,10-methylene-(6R)-tetrahydrofolic acid, which, as a co-factor, forms a cytostatically active, covalent ternary complex: 5-F-dUMP/TS/methylenetetrahydrofolic acid with 5-fluorodeoxyuridine monophosphate (5-F-dUMP) and thymidylate synthetase (TS) formed from 5-fluorouracil (5-FU). See W. A. Bleyer, Cancer, Supplement, March 15, 1989, pp. 995-1007, and E. L. R. Stokstad, Folic Acid Metabolism in Haltha nd Disease 1990, Wiley-Liss, Inc., p. 9.
It would, therefore, be advantageous to use the cofactor 5,10-methylenetetrahydrofolic acid directly instead of leucovorin (5-formyl-tetrahydrofolic acid). Until now, this undertaking has failed because 5,10-methylenetetrahydrofolic acid and its salts have been unstable and generally of insufficient purity. For example, recrystallization has not been successful because the known salts decompose during recrystallization, resulting in little or no improvement in purity.
Such known salts of 5,10-methylenetetrahydrofolic acid--mainly alkali-, alkaline-earth, and alkanolamine salts--have been described in various publications. For example, B. T. Kaufmann et al.., J. Biol. Chem. 238, 1498-1500 (1963), described a "relatively pure isomer of 5,10-methylenetetrahydrofolic acid" as alkali salt, confirmed by V. F. Scott et al. in Biochem. and Biophys. Res. Comm. 14, 523-526 (1964). An alkanolamine salt (Tris) with a purity of more than 97% was described by J. E. Wright et al., Cancer Research 49, 2592-2596 (1989). Also, the precipitation of 5,10-methylenetetrahydrofolic acid by the addition of DCl and the following transformation into the sodium salt was reported in M. Poe et al., Biochemistry 18 (24), 5527-5528 (1979). The barium salt of 5,10-methylenetetrahydrofolic acid was disclosed by R. L. Blakey et al. in The Journal of Biological Chemistry 238 (9), 3075-3079 (1963). See also EP-A-0 409 125.